This invention relates to a composition for examining urobilinogen in the urine.
Urobilinogen is produced from the bile appeared in the intestines by reductive action of bacteria. A part of the urobilinogen is absorbed by the portal vein and passed to the liver, while almost all the urobilinogen is ingested by liver cells, excreted in the bile as it is, and led to the intestines. But a part of it passes through the liver and enters into the systemic circulation, and further excreted in the urine.
The amount of urobilinogen in the urine increases in the case of liver function disorder, hemolytic diseases and ileus. Therefore, the examination of urobilinogen in the urine is one of important clinical examinations in the diagnosis of these diseases.
As methods for measuring urobilinogen, there has widely been used a method of using a test paper wherein there is used the condensation reaction between p-dimethylaminobenzaldehyde and urobilinogen to produce a reddish violet color. But this method had defects in that (1) the color-producing reaction is slow and it requires 1 minute or more for the determination, (2) it lacks in specificity and false positive reactions take place by the presence of porphobillinogen, indole, skatole, etc., and (3) a large amount of urea present in the urine reacts with p-dimethylaminobenzaldehyde to produce a yellow color which interfers the determination.
In order to remove such defects, there are reported various color-producing methods using diazonium salts which are faster in the reaction rate for urobilinogen and higher in specificity than p-dimethylaminobenzaldehyde. Generally speaking, diazonium salts also react with bilirubin, but it is possible to measure urobilinogen without influenced by bilirubin by applying the difference in electron affinity of the diazonium salts. For example, it is proposed to use a benzenediazonium salt wherein at least one substituent at the ortho or para position of a phenyl group is a multiatomic electron donating group having at least one mesomeric electron pair and the total of Hammet's .sigma. values of all the substituents is not exceeding +0.4 (Ger. Offen. 2,130,559, Chem. Abstr. 78, 1973, 156279u). It is also proposed to use a diazonium salt selected from phenyl-, pyrrole- and pyrazolediazonium salts that are eventually fused or directly substituted with iso- or heterocyclic aromatic compounds in a mesomeric-like and sterically unhindered position, as well as pyridine- and pyrazoldiazonium salts that in a mesomeric position at least one polyatomic electron donar group with at least one measomeric active pair of electrons, the total of Hammet's .sigma. values of all substituents and hetero-atoms not exceeding +0.6 (Ger. Offen. 2,229,611, Chem. Abstr. 80, 1974, 142770b). It is further proposed to use 4-fluoro-3-nitrobenzenediazonium salts (U.S. Pat. No. 3,989,462) and anthraquinone series diazonium salts (Japanese Patent Appln. Kokai (Laid-Open) No. 114954/80, Chem. Abstr. 94:43712S, 1981). But these diazonium salts are low in sensitivity for color production with urobilinogen (molecular extinction coefficient: .epsilon.= 8,000-17,000) and since the color produced is brown to orange in many cases, it is difficult to determine when the urine is colored darkly.